Arbuscular mycorrhizal fungi spore propagation using single spore as starter inoculum and a plant host.

AIMS: The propagation of pure cultures of arbuscular mycorrhizal fungal (AMF) is an essential requirement for their large-scale agricultural application and commercialization as biofertilizers. The present study aimed to propagate AMF using the single-spore inoculation technique and compare their propagation ability with the known reference spores. METHODS AND RESULTS: Arbuscular mycorrhizal fungal spores were collected from salt-affected Saemangeum reclaimed soil in South Korea. The technique involved inoculation of sorghum-sudangrass (Sorghum bicolor L.) seedlings with single, healthy spores on filter paper followed by the transfer of successfully colonized seedlings to 1-kg capacity pots containing sterilized soil. After the first plant cycle, the contents were transferred to 2·5-kg capacity pots containing sterilized soil. Among the 150 inoculated seedlings, only 27 seedlings were colonized by AMF spores. After 240 days, among the 27 seedlings, five inoculants resulted in the production of over 500 spores. The 18S rDNA sequencing of spores revealed that the spores produced through single-spore inoculation method belonged to Gigaspora margarita, Claroideoglomus lamellosum and Funneliformis mosseae. Furthermore, indigenous spore F. mosseae M-1 reported a higher spore count than the reference spores. CONCLUSIONS: The AMF spores produced using the single-spore inoculation technique may serve as potential bio-inoculants with an advantage of being more readily adopted by farmers due to the lack of requirement of a skilled technique in spore propagation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the current study describe the feasible and cost-effective method to mass produce AMF spores for large-scale application. The AMF spores obtained from this method can effectively colonize plant roots and may be easily introduced to the new environment.

Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria.

Biotic stress like pathogenic infection increases ethylene biosynthesis in plants and ethylene inhibitors are known to alleviate the severity of plant disease incidence. This study aimed to reduce the bacterial spot disease incidence in tomato plants caused by Xanthomonas campestris pv. vesicatoria (XCV) by modulating stress ethylene with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Methylobacterium strains. Under greenhouse condition, Methylobacterium strains inoculated and pathogen challenged tomato plants had low ethylene emission compared to pathogen infected ones. ACC accumulation and ACC oxidase (ACO) activity with ACO related gene expression increased in XCV infected tomato plants over Methylobacterium strains inoculated plants. Among the Methylobacterium spp., CBMB12 resulted lowest ACO related gene expression (1.46 Normalized Fold Expression), whereas CBMB20 had high gene expression (3.42 Normalized Fold Expression) in pathogen challenged tomato. But a significant increase in ACO gene expression (7.09 Normalized Fold Expression) was observed in the bacterial pathogen infected plants. In contrast, Methylobacterium strains enhanced ß-1,3-glucanase and phenylalanine ammonia-lyase (PAL) enzyme activities in pathogen challenged tomato plants. The respective increase in ß-1,3-glucanase related gene expressions due to CBMB12, CBMB15, and CBMB20 strains were 66.3, 25.5 and 10.4% higher over pathogen infected plants. Similarly, PAL gene expression was high with 0.67 and 0.30 Normalized Fold Expression, in pathogen challenged tomato plants inoculated with CBMB12 and CBMB15 strains. The results suggest that ethylene is a crucial factor in bacterial spot disease incidence and that methylobacteria with ACC deaminase activity can reduce the disease severity with ultimate pathogenesis-related protein increase in tomato.

Characterization of plant growth-promoting traits of bacteria isolated from larval guts of diamondback moth Plutella xylostella (lepidoptera: plutellidae).

Eight bacterial isolates from the larval guts of Diamondback moths (Plutella xylostella) were tested for their plant growth-promoting (PGP) traits and effects on early plant growth. All of the strains tested positive for nitrogen fixation and indole 3-acetic acid (IAA) and salicylic acid production but negative for hydrogen cyanide and pectinase production. In addition, five of the isolates exhibited significant levels of tricalcium phosphate and zinc oxide solubilization; six isolates were able to oxidize sulfur in growth media; and four isolates tested positive for chitinase and beta-1,3-glucanase activities. Based on their IAA production, six strains including four that were 1-aminocyclopropane-1-carboxylate (ACC) deaminase positive and two that were ACC deaminase negative were tested for PGP activity on the early growth of canola and tomato seeds under gnotobiotic conditions. Acinetobacter sp. PSGB04 significantly increased root length (41%), seedling vigor, and dry biomass (30%) of the canola test plants, whereas Pseudomonas sp. PRGB06 inhibited the mycelial growth of Botrytis cinerea, Colletotrichum coccodes, C. gleospoiroides, Rhizoctonia solani, and Sclerotia sclerotiorum under in vitro conditions. A significant increase, greater than that of the control, was also noted for growth parameters of the tomato test plants when the seeds were treated with PRGB06. Therefore, the results of the present study suggest that bacteria associated with insect larval guts possess PGP traits and positively influence plant growth. Therefore, insect gut bacteria as effective PGP agents represent an unexplored niche and may broaden the spectrum of beneficial bacteria available for crop production.

Ecological occurrence of Gluconacetobacter diazotrophicus and nitrogen-fixing Acetobacteraceae members: their possible role in plant growth promotion.

Gluconacetobacter diazotrophicus has a long-standing history of bacterial-plant interrelationship as a symbiotic endophyte capable of fixing atmospheric nitrogen. In low nitrogen fertilized sugarcane fields it plays a significant role and its occurrence was realised in most of the sugarcane growing countries. In this mini review, the association of G. diazotrophicus with sugarcane, other crop plants and with various hosts is discussed. The factors affecting survival in the rhizosphere and the putative soil mode of transmission are emphasized. In addition, other N(2)-fixing Acetobacteraceae members, including Gluconacetobacter azotocaptans, Gluconacetobacter johannae and Swaminathania salitolerans, occurring in coffee, corn and rice plants are also covered. Lastly, the plant-growth-promoting traits identified in this group of bacteria, including N(2) fixation, phytohormone synthesis, P and Zn solubilization and biocontrol, are analysed.

Energy status and functioning of phosphorus-deficient soybean nodules.

Characterization of the effects of long-term P deficiency and of onset and recovery from P deficiency on bacteroid mass and number per unit nodule mass and energy status of soybean (Glycine max L. Merr.) nodules was used to investigate the mechanisms by which P deficiency decreases symbiotic N(2) fixation. The continuous P deficiency treatment (0.05 millimolar P) significantly decreased the whole plant dry mass, P, and N by 62, 90, and 78%, respectively, relative to the P-sufficient control (1.0 millimolar) at 44 days after transplanting. Specific nitrogenase activity was decreased an average of 28% over a 16-day experimental period by P deficiency. Whole nodules of P-deficient controls contained 70 to 75% lower ATP concentrations than nodules of P-sufficient controls. Energy charge and ATP concentrations in the bacteroid fraction of nodules were not significantly affected by P treatment. However, ATP and total adenylate concentrations and energy charge in the plant cell fraction of nodules were significantly decreased 91, 62, and 50%, respectively, by the P deficiency treatment. Specific nitrogenase activity, energy charge, and ATP concentration in the plant cell fraction increased to the levels of nonstressed controls within 2, 2, and 4 days, respectively, after alleviation of external P limitation, whereas bacteroid mass per unit nodule mass and bacteroid N concentration did not increase to the level of nonstressed controls until 7 days after alleviation of external P limitation. All of these parameters except bacteroid mass per unit nodule mass decreased to the levels of the P-deficient controls by 11 days after onset of external P limitation. Concentration of ATP in the bacteroid fraction was not significantly affected by alteration in the external P supply. Energy charge in the bacteroid fraction from plants recovering from P deficiency was decreased to a small (10%) but significant extent (P < 0.05) at two sampling dates relative to P-sufficient controls. These ATP concentration and energy charge measurements indicate that P deficiency impaired oxidative phosphorylation in the plant cell fraction of nodules to a much greater extent than in the bacteroids. The concurrence of significant changes in specific nitrogenase activity (2 days) and in the energy charge (2 days) and ATP concentration (4 days) in the plant cell fraction during recovery from external P limitation is consistent with the conclusion that P deficiency decreases the specific nitrogenase activity by inhibiting an energy-dependent reaction(s) in the plant cell fraction of the nodules.